定点突变技术提高β-葡萄糖苷酶活性  

Improving β-glucosidase Activity by Site-directed Mutagenesis

作　　者：李沛华[1] 郭雪睿 彭继燕 陈惠[1] 唐自钟[1] 金伟琼[1] 王欢[1] 韩学易[1] 

机构地区：[1]四川农业大学,生命科学学院,雅安625014

出　　处：《基因组学与应用生物学》2016年第8期2083-2091,共9页Genomics and Applied Biology

基　　金：国家级大学生创新训练计划项目(项目号1510626004)资助

摘　　要：β-葡萄糖苷酶(Bgl)是纤维素水解过程中的限速关键酶。提高Bgl的酶活,对于增强纤维素酶水解力有着十分重要的作用。本研究根据3D同源模建及分子对接,预测分析活性中心,利用定点突变做出针对性的改造。对来源于米曲霉的β-葡萄糖苷酶基因进行六个位点的定点突变,并将其在大肠杆菌中进行了突变基因的高效表达。经IPTG诱导后,分离纯化并进行酶学性质分析,获得酶活力提高的两个突变位点(Asn^(347)Ser,Gly^(235)Met),结果表明N^(347)S和G^(235)M位点的突变使酶的比活力显著提高,比原始酶活分别提高了43.1%和14.7%。这一研究为进一步提高β-葡萄糖苷酶活性研究提供一定的理论依据。β-glucosidase （Bgl） is the key enzyme in the hydrolysis of cellulose. Improving the enzyme activity of Bgl plays a critical role in enhancing the hydrolysis of cellulose. The site directed mutagenesis of six sites from Aspergillus niger was carried out, and the gene was expressed in Escherichia coll. This study was based on 3D homology modeling and molecular docking, predicted and analyzed Bgl active center, afterwards used site-directed mutagenesis to make targeted transformation. After IPTG induction, purification and enzymatic properties analysis, obtained two site-mutations （Asn347Ser, Gly235Met） which enhanced the activity of Bgl. Results showed N347S and G235M site mutations significantly increased the activity of Bgl 43.1% and 14.7% higher, respectively. This study laid a certain foundation for further improvement of the activity of β-glucosidase.

关 键 词：Β-葡萄糖苷酶 定点突变 酶学性质 大肠杆菌 

分 类 号：Q55[生物学—生物化学]